Trimethyl histone h3 lys 9

Lentiviral pLV vector expressing Flag-KMT1A [38 (link)] and LV-HA-MKK6EE and pLV-HA-MKK6DN were generated by subcloning inserts from pcDNA-HA-MKK6EE and pcDNA-HA-MKK6DN (provided by Dr. L. Puri) [39 (link)] into pLV vector. For expression of shRNA, KMT1A, p38α, or scramble shRNAs are cloned individually into lentiviral pLKO.1-TRC vector (Addgene) and sequence verified. The shRNA sequences for KMT1A and scramble were described previously [38 (link)]. The sequence for p38α shRNA was 5′-AGCCCAGCAACCTAGCTGTTT-3′. Vectors pGEX-4T-3-H3(N) [26 (link)] and pGEX-ATF2 (provided by Dr. J. Han) [40 (link)] express GST fusion N-terminal histone H3 and ATF2 proteins, respectively.
Antibodies used were phospho-p38 (Cell Signaling 9215), β-actin-peroxidase (Sigma A3854), Flag-M2 (Sigma F3165), myogenin (BD Pharmingen 556358), KMT1A (Cell Signaling 8729, and Millipore 07-550 and 05-615), MyoD (Santa Cruz sc-760 and BD Pharmingen 554130), p38α (Cell Signaling 9790), HA-peroxidase (Sigma H6533), acetyl-histone H3 (Millipore, 06-599), trimethyl-histone H3 (Lys-9) (Millipore 07-442), trimethyl-histone H3 (Lys27) (Millipore 07-449), GAPDH (Biodesign H86504M), Brg-1 (Santa Cruz sc-10768), p21^cip1 (Santa Cruz sc-397), myosin heavy chain (Developmental Studies Hybridoma Bank, MF-20), total p38 (Cell Signaling 9212), and normal rabbit IgG (Santa Cruz sc-2027).

Protein preparation and Western blotting were performed according to the method previously described.44 The mitochondrial fraction was prepared using the Mitochondria Isolation Kit (cat#89874; Thermo Scientific, Rockford, IL, USA) according to the instructions provided. Protein concentration was determined using the BCA Assay Reagent (cat#23228; Pierce, Rockford, IL, USA). Western blotting was performed as previously described.44 Primary antibodies were used for immunoblotting: Bmi1 (#6964), Mcl‐1 (#5453), Bcl‐2 (#2870), Bcl‐xL (#2764), cleaved caspase‐3 (#9664), cleaved caspase‐9 (#9505), cleaved PARP (#5625), and Tri‐Methyl‐Histone H3 (Lys27) (#9733) from Cell Signaling Technology (Danvers, MA, USA); Tri‐Methyl‐Histone H3 (Lys9) (#07‐) from Millipore (Burlington, MA, USA); Noxa (#OP180) from Merck Millipore (Darmstadt, Germany); p53 (sc‐126) from Santa Cruz Biotechnology (Dallas, TX, USA); β‐actin (A5316) from Sigma‐Aldrich (St. Louis, MO, USA). Secondary antibodies were anti‐rabbit IgG HRP (#7074) and anti‐mouse IgG HRP (#7076) and purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody conjugates were visualized by chemiluminescence (ECL; cat#34076; Thermo, Rockford, IL, USA).

ChIP analysis of YG8JR and Y47JR mouse tissues (n = 4) was carried out by initial cross-linking of DNA and protein by formaldehyde treatment of homogenized cerebellum tissue samples (45 mg each). DNA was then sheared by sonication using a Bioruptor Pico sonication system (Diagenode), followed by immunoprecipitation with trimethyl-Histone H3 (Lys9) (Millipore Cat# 07-442, RRID:AB_310620) or acetyl-Histone H3 (Lys9) (Millipore Cat# 07-352, RRID:AB_310544) antibodies. The DNA was reverse-cross linked and extracted by standard phenol/chloroform/glycogen extraction and ethanol precipitation. Quantitative PCR amplification of the co-immunoprecipitated DNA was carried out with Fast SYBR Green in a QuantStudio 7 Flex Real-Time PCR (Applied Biosystems, MA, United States) using three sets of FXN primers (Pro: promoter, Up: upstream, Down: downstream of GAA repeat region) as previously described (Herman et al., 2006 (link); Al-Mahdawi et al., 2008 (link)). Each value of immunoprecipitated DNA was processed in triplicate qPCR analysis. Relative quantification values were identified by 2^–ΔΔCt method and were normalized to the input values. For each experiment, minus antibody immunoprecipitation and ddH₂O without chromatin samples were used as negative controls.